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Rational improvement of the engineered isobutanol-producing Bacillus subtilis by elementary mode analysis.

Li S, Huang D, Li Y, Wen J, Jia X - Microb. Cell Fact. (2012)

Bottom Line: Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis.The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

ABSTRACT

Background: Isobutanol is considered as a leading candidate for the replacement of current fossil fuels, and expected to be produced biotechnologically. Owing to the valuable features, Bacillus subtilis has been engineered as an isobutanol producer, whereas it needs to be further optimized for more efficient production. Since elementary mode analysis (EMA) is a powerful tool for systematical analysis of metabolic network structures and cell metabolism, it might be of great importance in the rational strain improvement.

Results: Metabolic network of the isobutanol-producing B. subtilis BSUL03 was first constructed for EMA. Considering the actual cellular physiological state, 239 elementary modes (EMs) were screened from total 11,342 EMs for potential target prediction. On this basis, lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHC) were predicted as the most promising inactivation candidates according to flux flexibility analysis and intracellular flux distribution simulation. Then, the in silico designed mutants were experimentally constructed. The maximal isobutanol yield of the LDH- and PDHC-deficient strain BSUL05 reached 61% of the theoretical value to 0.36 ± 0.02 C-mol isobutanol/C-mol glucose, which was 2.3-fold of BSUL03. Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.

Conclusions: EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis. The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification. This network-based rational strain improvement strategy could serve as a promising concept to engineer efficient B. subtilis hosts for isobutanol, as well as other valuable products.

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Related in: MedlinePlus

EMs for isobutanol and biomass biosynthesis in isobutanol-producing B. subtilis BSUL03. The solution of the EMs is represented by the blue circles. All possible solutions locate the interior as well as the sides of the rectangular triangle. EMs on the axes represent extreme modes exclusively linked to the formation of either isobutanol or biomass. The solid triangles indicate the best modes of the simulated mutants at optimal performance. The hollow triangles indicate the experimental values of different mutants in batch fermentations. Red, BSUL03; yellow, BSUΔldh or BSUL04; green, BSUΔldhΔpdhC or BSUL05. The pink pentagram indicates the experimental values of BSUL05 in fed-batch fermentations.
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Figure 1: EMs for isobutanol and biomass biosynthesis in isobutanol-producing B. subtilis BSUL03. The solution of the EMs is represented by the blue circles. All possible solutions locate the interior as well as the sides of the rectangular triangle. EMs on the axes represent extreme modes exclusively linked to the formation of either isobutanol or biomass. The solid triangles indicate the best modes of the simulated mutants at optimal performance. The hollow triangles indicate the experimental values of different mutants in batch fermentations. Red, BSUL03; yellow, BSUΔldh or BSUL04; green, BSUΔldhΔpdhC or BSUL05. The pink pentagram indicates the experimental values of BSUL05 in fed-batch fermentations.

Mentions: Metabolic network of the isobutanol-producing B. subtilis BSUL03 for EMA comprises 131 reactions (36 reversible and 95 irreversible) and 132 metabolites (Additional file 1, Table S1 and Table S2). Overall, this metabolic network was decomposed into a total of 11,342 elementary modes (EMs). Each mode represents a unique possible pathway with balanced metabolites and cofactors. Among all the EMs, the majority are extreme modes exclusively linked to the production of either biomass or isobutanol, locating on the two axes of the plot (Figure 1). About 0.2% of the total EMs (25 EMs) allowed the maximal theoretical isobutanol yield of 0.67 C-mol isobutanol/C-mol glucose (C-mol/C-mol), all of them had no biomass and byproduct. Conversely, only EMs without isobutanol biosynthesis could reach the maximal biomass yield of 0.52 C-mol biomass/C-mol glucose, close to the value of the wild type B. subtilis[25]. Considering the coupled formation of isobutanol and biomass, the total EMs were constrained to 2,216 EMs (less than 20% of the total EMs) with the maximal theoretical isobutanol yield of 0.64 C-mol/C-mol. The large number of EMs illustrates the robustness and flexibility of the cells to adapt themselves to particular conditions by using different pathways.


Rational improvement of the engineered isobutanol-producing Bacillus subtilis by elementary mode analysis.

Li S, Huang D, Li Y, Wen J, Jia X - Microb. Cell Fact. (2012)

EMs for isobutanol and biomass biosynthesis in isobutanol-producing B. subtilis BSUL03. The solution of the EMs is represented by the blue circles. All possible solutions locate the interior as well as the sides of the rectangular triangle. EMs on the axes represent extreme modes exclusively linked to the formation of either isobutanol or biomass. The solid triangles indicate the best modes of the simulated mutants at optimal performance. The hollow triangles indicate the experimental values of different mutants in batch fermentations. Red, BSUL03; yellow, BSUΔldh or BSUL04; green, BSUΔldhΔpdhC or BSUL05. The pink pentagram indicates the experimental values of BSUL05 in fed-batch fermentations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475101&req=5

Figure 1: EMs for isobutanol and biomass biosynthesis in isobutanol-producing B. subtilis BSUL03. The solution of the EMs is represented by the blue circles. All possible solutions locate the interior as well as the sides of the rectangular triangle. EMs on the axes represent extreme modes exclusively linked to the formation of either isobutanol or biomass. The solid triangles indicate the best modes of the simulated mutants at optimal performance. The hollow triangles indicate the experimental values of different mutants in batch fermentations. Red, BSUL03; yellow, BSUΔldh or BSUL04; green, BSUΔldhΔpdhC or BSUL05. The pink pentagram indicates the experimental values of BSUL05 in fed-batch fermentations.
Mentions: Metabolic network of the isobutanol-producing B. subtilis BSUL03 for EMA comprises 131 reactions (36 reversible and 95 irreversible) and 132 metabolites (Additional file 1, Table S1 and Table S2). Overall, this metabolic network was decomposed into a total of 11,342 elementary modes (EMs). Each mode represents a unique possible pathway with balanced metabolites and cofactors. Among all the EMs, the majority are extreme modes exclusively linked to the production of either biomass or isobutanol, locating on the two axes of the plot (Figure 1). About 0.2% of the total EMs (25 EMs) allowed the maximal theoretical isobutanol yield of 0.67 C-mol isobutanol/C-mol glucose (C-mol/C-mol), all of them had no biomass and byproduct. Conversely, only EMs without isobutanol biosynthesis could reach the maximal biomass yield of 0.52 C-mol biomass/C-mol glucose, close to the value of the wild type B. subtilis[25]. Considering the coupled formation of isobutanol and biomass, the total EMs were constrained to 2,216 EMs (less than 20% of the total EMs) with the maximal theoretical isobutanol yield of 0.64 C-mol/C-mol. The large number of EMs illustrates the robustness and flexibility of the cells to adapt themselves to particular conditions by using different pathways.

Bottom Line: Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis.The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

ABSTRACT

Background: Isobutanol is considered as a leading candidate for the replacement of current fossil fuels, and expected to be produced biotechnologically. Owing to the valuable features, Bacillus subtilis has been engineered as an isobutanol producer, whereas it needs to be further optimized for more efficient production. Since elementary mode analysis (EMA) is a powerful tool for systematical analysis of metabolic network structures and cell metabolism, it might be of great importance in the rational strain improvement.

Results: Metabolic network of the isobutanol-producing B. subtilis BSUL03 was first constructed for EMA. Considering the actual cellular physiological state, 239 elementary modes (EMs) were screened from total 11,342 EMs for potential target prediction. On this basis, lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHC) were predicted as the most promising inactivation candidates according to flux flexibility analysis and intracellular flux distribution simulation. Then, the in silico designed mutants were experimentally constructed. The maximal isobutanol yield of the LDH- and PDHC-deficient strain BSUL05 reached 61% of the theoretical value to 0.36 ± 0.02 C-mol isobutanol/C-mol glucose, which was 2.3-fold of BSUL03. Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.

Conclusions: EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis. The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification. This network-based rational strain improvement strategy could serve as a promising concept to engineer efficient B. subtilis hosts for isobutanol, as well as other valuable products.

Show MeSH
Related in: MedlinePlus